1. Field of the Invention
The present invention relates generally to immunologic measurement systems, in which antibody binding is used to measure substance concentration levels, More particularly, the present invention relates to an electrochemical immunosensor in which voltametric signals are used to convert immunosensor binding events to a measurable electrical quantity.
2. Description of Related Art
Immunosensors are a subset of the class of biological measurement instruments commonly known as biosensors. Biosensors typically consist of probes containing biological recognition molecules. The recognition molecules respond to the presence of certain substances, and the response is then converted to a measurable quantity. Biosensors thus provide an indication of substance concentration without the use of complicated laboratory procedures. An immunosensor is a particular type of biosensor in which an antibody serves as the biological recognition molecule. Antibodies are produced in the human body to inactivate foreign substances by irreversibly combining with or binding the substance to form a complex. An almost unlimited variety of antibodies are produced, each specific to a particular substance. The term "antigen" as used herein refers to any substance that can cause the immune system to form antibodies. The term "analyte" will be used to refer to any substance, including an antigen, which binds with any other substance to form a complex. Substances which bind analytes will be referred to as "binding agents". An antibody is one particular type of binding agent. Other binding agents include receptors and affinity-binding molecules.
The highly sensitive and selective nature of antibody binding permits the immunosensor to accurately detect minute analyte concentration levels. Immunsensors are therefore useful in medical applications involving the detection of hormones, illegal drugs or other trace constituents present in blood and urine. Furthermore, since antibodies can be selected to bind to a wide variety of different analytes, the immunosensor has the potential for widespread application to many non-medical uses such as industrial and food processing quality control, monitoring of environmental pollutants and detection of materials used in chemical and biological warfare.
Despite these advantages and potentially widespread applications, immunosensors are not commercially available at the present time. A significant obstacle has been the problem of accurately converting the antibody recognition event to a measurable signal. As a result, more complex measurement techniques are currently used in place of immunosensors. One such group of techniques involves multi-step analytic laboratory procedures known as immunoassays. Several exemplary immunoassays are described in E. Harlow and. D. Lane, Antibodies: a Laboratory Manual, pp. 553-612. In applications requiring a highly accurate measurement, the immunoassay techniques used typically require highly trained technicians and costly special equipment. The radioimmunoassay ("RIA") is a good example of this type of highly accurate immunoassay. Measurements are therefore typically performed at a fully equipped central laboratory rather than at remote user locations such as a medical office or a factory. The complex conversion process results in a significant delay before availability of measurement results.
In applications in which a lower degree of accuracy is acceptable, immunoassay techniques which make use of subjective visual examination are typically used. Such techniques provide a convenient but less accurate screen in variety of situations. The subjective tests are often of the "dipstick" type. A sensitized material is placed in the analyte, and a color change on the surface of the material occurs. The pregnancy tests available in most supermarkets are usually of this type. This type of test generally belongs to a class of immunoassays known as enzyme linked immunosorbent assay ("ELISA"). Although ELISA techniques can provide fast results, they are intrinsically less accurate than radioimmunoassay techniques.
There are additional problems with particular immunoassay techniques. For example, one of the most sensitive of the immunoassays is the two-step sandwich type immunoradiometric assay ("IRMA") in which the complexation of an antibody with an antigen is detected by measuring the amount of radioactivity emitted by a radiolabeled antibody. The use of radioactive labels contributes to sensitivity, but exposes laboratory personnel to a significant safety hazard. Moreover, radioactive compounds have a limited shelf life. Other immunoassay techniques, such as fluorescence polarization immunoassay ("FPIA") and ELISA, are safer for laboratory personnel and more stable with regard to shelf life, but are generally less sensitive than the IRMA.
Another alternative to electrochemical immunosensors uses light scattering to convert the antibody binding response to a measurable electrical signal. One such technique uses a spectrometer to measure the variation in the spectrum of laser light passing through a solution containing antibodies and antigens. The antibody-antigen binding response alters the spectral characteristics of the laser in accordance with the antigen concentration level. U.S. Pat. No. 4,725,149 is a variation on this general technique in which laser light is passed through a solution containing antigens and magnetic particles coated with antibodies. The magnetic particles are rotated at a particular frequency by signals applied to coils surrounding the solution such that antibody-antigen binding events produce a measurable variation in the scattered light. In U.S. Pat. No. 4,762,413 a photodetector measures the power spectral density of fluctuations in scattered light intensity, and a mean power spectral density value taken over several measurements is used to indicate antigen concentration.
Light scattering conversion techniques such as these, however, typically utilize expensive specially designed equipment which would tend to limit use at remote sites. In addition, the techniques generally require combining the measurement sample with a buffer solution containing a quantity of particles. The antibodies which will bind to the antigens in the sample are fixed to the surfaces of the particles. Thus, the light scattering techniques are not readily adaptable to those situations in which a sample cannot be easily removed and combined with the particle solution. Furthermore, since the recognition molecules are placed within the particle solution rather than on a probe, the light scattering techniques do not share the advantages of convenience and simplicity commonly associated with biosensors.
Attempts have been made to simplify optical immunosensor detection systems such as those discussed above by attaching antibodies or other binding agents to a deformable organic polymer film. The polymer film absorbs green light and fluoresces strongly in the orange part of the spectrum. When an antibody attached to a surface of the film combines with an analyte, the polymer film is perturbed, causing a detectable decrease in the fluorescence of the film at the point of the combination. The decrease in fluorescence is proportional to the number of analyte molecules bound to antibodies on the surface of the film. The amount of light reflected from the film is measured to determine the analyte concentration in a particular sample. See "Signal Transduction on Film" in Bioventure View, March 1992. Although the polymer film approach may decrease the complexity of the optical immunosensor, it presents additional problems. One such problem is the inability to place a sufficient quantity of binding agents at particular points on the film.
As is apparent from the above, there presently is a need for a simple and inexpensive immunosensor system in which antibody recognition events are rapidly and accurately converted to readily monitored electrical signals using standard electronic test equipment. The immunosensor system should provide highly sensitive, selective and repeatable measurements without the problems associated with immunoassay or light scattering techniques. Furthermore, the immunosensor and related equipment should be easily adapted to the specific requirements of a variety of different uses, including but not limited to medical, industrial, environmental and military applications.